The Mg-containing (bacterio)chlorophylls ((B)Chl) and their free bases, the (bacterio)pheophytins ((B)Phe), are essential to photosynthesis. They act as antenna or redox pigments enabling light-induced charge separation within the reaction center. The pigments are also potentially useful photosensitizers, e.g. in photodynamic tumor therapy.
Porphyrins have been shown to accumulate in tumor tissue and, upon irradiation of the tumor tissue, to absorb light in situ, providing a mean to detect tumors by location of the fluorescence. A crude derivative of hematoporphyrin, known as hematoporphyrin derivative or HPD, has been proposed both for detection and for photodynamic therapy of tumors. A form of HPD said to be more effective comprises a portion of HPD having an aggregate weight over 10 Kda and is the subject of U.S. Pat. No. 4,649,151. HPD or its active components have been described in U.S. Pat. No. 4,753,958 for topical treatment of skin diseases, and in Matthews et al., 1988, for sterilization of biological samples containing infectious organisms such as bacteria and virus.
In order to optimize the performance of the porphyrin drugs in therapeutics and diagnostics, several porphyrin derivatives have been proposed in which, for example, there is a central metal atom complexed to the four pyrrole rings, and/or the peripheral substituents of the pyrrole rings are modified and/or the macrocycle is dihydrogenated to Chl derivatives (chlorins) or tetrahydrogenated to BChl derivatives (bacteriochlorins).
Complexes of cyclic tetrapyrroles with metals other than Mg were studied in the porphyrin and 17,18-dihydroporphyrin series to understand their spectrocospic and redox properties (Hynninen, 1991). Bacteriochlorophylls are of potential advantage compared to the chlorophylls because they show intense near-infrared bands, i.e. at considerably longer wavelengths than chlorophyll derivatives. However, little information is presently available on bacteriochlorophylls with central metals other than Mg.
PCT International Application Publication No. WO 90/12573 to Dougherty describes derivatives of bacteriochlorophyll-a or -b or of the corresponding bacteriochlorins devoid of the central metal atom or in which the central metal atom may be a non-paramagnetic metal selected from Mg.sup.2+, Sn.sup.2+ and Zn.sup.2+, and the C-17.sup.3 -carboxyl group is esterified with a saturated or unsaturated hydrocarbyl residue of 8-25C, for the manufacture of a composition for use in a method to effect the destruction or impairment of undesired target biological substrates, which method comprises photosensitizing said substrate with an effective amount of said derivative, followed by irradiation of the target substrate with radiation in a wavelength band absorbed by said derivative for a time effective to impair or destroy the substrate. In addition, the compounds are said to be useful in photodynamic therapy and diagnostics. It is to be noted that although Sn.sup.2+ and Zn.sup.2+ complexes of bacteriochlorophyll-a or -b are claimed, these metal derivatives have not been exemplified nor was any method for their preparation described in the specification of said patent application WO 90/12573.
Losev et al, 1990, describe [Pd]--BChl and [Cu]--BChl complexes said to be prepared by direct metalation of BPhe with Pd benzonitrile in benzene in a stream of nitrogen or with a concentrated solution of CuCl.sub.2 in methanol, respectively. However, this publication lacks details of the method of preparation and characterization of the metal complexes. Moreover, the preparation of the [Pd]--BChl complex according to Losev could not be repeated by us.
Under normal delivery conditions, i.e. in the presence of oxygen at room temperature and under normal light conditions, the BChl moieties are labile and have somewhat lower quantum yields for triplet state formation, when compared with, e.g., hematoporphyrin derivative (HPD). However, their possible initiation of biological redox reactions, favorable spectral characteristics and their ready degradation in vivo result in the potential superiority of bacteriochlorophylls over other compounds, e.g. porphyrins and chlorophylls, for PDT therapy and diagnostics and for killing of cells, viruses and bacteria in samples and in living tissue. Chemical modification of bacteriochlorophylls is expected to further improve their properties, but this has been very limited due to lack of suitable methods for the preparation of such modified bacteriochlorophylls (Hynninen, 1991).
European Patent Application published under No. 0584552 of the same applicant of the present application describes new conjugates of Chl and BChl with amino acids, peptides and proteins for use in PDT therapy and diagnostics. The amino acid, peptide or protein residue is linked directly or via a spacer to the C-17.sup.3 -carboxyl group of the Chl or BChl molecule. These conjugates are prepared by methods which are mild enough to retain the acid-labile central Mg atom. Zn and Cu complexes of chlorophyll a-17.sup.3 -serine methyl ester were also described therein, but no metalated bacteriochlorophyll nor a method for their preparation was described therein.
German Patent Application No. DE 4121876 describes bacteriochlorophyll derivatives in which modified esters at positions C-13.sup.2 and C-17.sup.3 are obtained under mild conditions by rapid alkaline transesterification, allowing further changes at the isocyclic ring while retaining the central Mg, by which the pigment absorption is shifted beyond 800 nm. The application also mentions metal complexes of said Bchl derivatives with Zn or Ni, but said complexes were not exemplified nor a method for their preparation was described therein.
It would be desirable to prepare new metalated complexes of BChl for use in PDT, in order to maintain or even improve the favorable optical and physiological properties of BChls while optimizing their photosensitizing potential as well as improving their chemical stability and optimizing their physiological lifetimes. Transmetalation results in distinct changes in the chemical reactivity and stability of the BChls, which are important for new modifications of the macrocycle and the peripheral substituents, and in particular for optimizing their transport, targeting and biological lifetime and minimizing toxic side effects. Transmetalation also results in distinct changes in the excited state properties, including triplet yield and lifetime, accessibility of higher excited states, and production of cytotoxic oxygen species.
Several methods are known for variation of the central metal atom in porphyrins (see Buchler, 1975). Porphyrins are readily accessible and chemically stable, yet spectrally and physiologically unfavorable.
Few methods are known for direct or indirect metalation of chlorophylls. Strell and Urumow, 1977, describe [Cr]--Chl and [Mn]--Chl complexes prepared by transmetalation of the [Cd]--Chl complex (obtained by reaction of the demetalated Chl derivative with cadmium acetate in methanol or pyridine) with the acetate of Cr.sup.++ or Mn in methanol under N.sub.2 atmosphere. This transmetalation method is said to be suitable also for Cu, Zn, Co and Pb complexes of chlorophyll derivatives, but not for Fe.sup.3+, Ni and Mg. However, since the Cu, Zn, Co and Pb complexes can be prepared by direct metalation into Phe, the method would be advantageous only for Cr and Mn. The authors also describe preparation of the [Mg]--Chl complex by direct metalation of Phe in acetone with Mg acetate in dimethylsulfoxide.
Little information is presently available on bacteriochlorophylls with central metals other than Mg. Metalation of bacteriochlorophylls is known to be more difficult than that of chlorophylls due to their decreased reactivity for metalation and increased reactivity for side reactions. A specific method for insertion of Mg into bacteriopheophytin a has been described (Wasielewsky, 1977). The present inventors have tried the direct metalation and transmetalation procedures for chlorophyll derivatives described by Strell and Urumow for the preparation of metal complexes of bacteriochlorophyll derivatives, but all attempts were unsuccessful. The direct metalation of bacteriopheophytin derivatives did not work with any metal tried, except for Cu and Zn, and resulted otherwise in a mixture of unreacted bacteriopheophytin and metalated oxidation products of the 3-acetyl-chlorophyll a type.